Phosphorylation of eukaryotic initiation factor -2 (eIF2) is an important mechanism regulating protein synthesis in response to different cellular stresses. This proposal focuses on one of these eIF2 kinases, GCN2, that regulates translation during amino acid limitation. In yeast, uncharged tRNA that accumulates during amino acid starvation combines with GCN2 sequences homologous to histidyl-tRNA synthetases (HisRS), inducing eIF2 kinase activity. Elevated phosphorylation of eIF2 stimulates the translational expression of GCN4, a transcriptional regulator of genes involved in different metabolic pathways. In addition to amino acid starvation, GCN4 translation is induced in response to glucose limitation, TOR Inhibition by rapamycin and exposure to elevated NaC1 concentrations. We will characterize the mechanisms activating GCN2 and study the integration of GCN4 translation in these stress pathways. Regulation of GCN2 also involves C-terminal sequences that are proposed to mediate multiple functions, including ribosome targeting and dimerization. We will address the importance of each of these functions in the regulation of GCN2. Interestingly, we found that the C-terminus of GCN2 can functionally replace the regulatory domain of the related eIF2 kinase, PKRI This so-called PKR chimera system will be exploited to identify diverse oligomerization and targeting domains and select for mutations to study the importance of these functions in the native protein. Given that starvation for amino acids stimulates eIF2 phosphorylation in mammalian cells, we searched for and identified a GCN2 homologue in mice. Like its yeast counterpart, mammalian GCN2 contains FlisRS-related sequences juxtaposed to the kinase catalytic domain. We will use GCN2 (+/+) and (-/-) mouse embryo fibroblast cells to study the regulation and function of GCN2I Furthermore, yeast and mammalian systems will be used to address whether the mechanism of leaky scanning important for GCN4 translation in yeast also function in response to eIF2 phosphorylation in mammals. Finally, will use the microarray hybridization assay to screen for genes that are induced in response to nutritional stress and assess the contribution of GCN2 activity. To pursue these fundamental questions, we propose four specific aims: 1) Characterization of the role of the C-terminus in activation of GCN2 in response to stress; 2) Characterization of the role of GCN2 protein kinase in multiple stress pathways in yeast; 3) Characterization of mechanisms regulating mammalian gene-specific translation by GCN2; and 4) Characterization of changes in gene expression in response to eIF2 phosphorylation by GCN2.